Contents

 Title
 Brief introduction about molecular biology tools

employed for human welfare

 Microbiome and microbiota and their role in
development of therapeutics
 Bacillus subtilis: Robust expression system !!Why
 Taxonomic profile of Bacillus subtilis
 Strains of Bacillus subtilis and molecules derived
 Literature review
 Exploration of selected species of Bacillus as
expression hosts for therapeutic proteins.
Brief introduction about molecular biology tools for human
welfare

 Molecular biology tools:

 E.coli : served as an important tool to study understand
primitive mechanism operative in lower organisms that
are relevant to humans as well in the construction of
vectors in r DNA technology, Sachharomyces
cerevisae(Yeast): Study of drug metabolizing enzymes and
metabolite profile. To understand many of the metabolic
activities that are conserved in humans.
Microbiome and microbiota and their role in development.

Microbiome is the genetic mapping of the microbes

that were helping humans right from human evolution
and protecting health.Microbiome provides us the ways
of endogenous therapeutic molecules that are produced
to maintain human health
 Microbiota is the billions of microbes inhabiting

human organs like skin,intestine, kidney etc.

Bacillus subtilis :why
 a.Non pathogenic and considered as GRAS(Generally Regarded as Safe)organism
 b.Can thrive in continuously changing environment due to its secretory enzymes like
proteases that can effectively degrade a variety of substrate.
 c.Excellent therapeutic protein secretion ability ,hence host for many therapeutic proteins
through rDNA technology. It has shorter fermentation cycle of around 48 hrs.(Years 180hrs)
 e.Excellent genetic expression system with good genetic stability and has no strong codon
preference.
 f.Feasibility of genetic manipulation of bacillus species through advanced tools like
CRISPR/cas9 and MEXI to reconstruct its cellular metabolic operations to Express
heterologous proteins(Metabolic engineering)
 g.Well understood transcription and translocation machinery to produce different
extracellular proteinsg. Ability to express genetically stable recombinant proteinsh. Novel
Bacillus strains with reduced nuclear and protease to produce proteins with commercial
viability
 h. At present 60%of the commercially available enzymes are produced through B.subtilis
B.subtilis strains .
 i.All the above advantages are possible as it has robust genomic sequence .K.Thus B.subtilis
is universal cell factory for industry, agriculture, bioengineered materials and therapeutic
arsenal.
Taxonomic profile
 Kingdom:Bacteria
 Sub kingdom: Posibacteria
 Phylum:Firmicutes
 Class:Bacilli
 Order: Bacillales
 Family: Bacillaceae
 Genus:Bacillus
 Species:Bacillus subtilis
Strains and Therapeutic Molecules
Produced
Literature Survey
 Lidia Westers et.al., studied on Bacillus subtilis as cell factory for
pharmaceutical proteinsBacillus subtilis as cell factory for pharmaceutical
proteins. Bacillus subtilis is a rod-shaped, Gram-positive soil bacterium that
secretes numerous enzymes to degrade a variety of substrates, enabling the
bacterium to survive in a continuously changing environment. These enzymes
are produced commercially and this production represents about 60% of the
industrial-enzyme market. Unfortunately, the secretion of heterologous proteins,
originating from Gram-negative bacteria or from eukaryotes, is often severely
hampered. Several bottlenecks in the B. subtilis secretion pathway, such as poor
targeting to the translocase, degradation of the secretory protein, and incorrect
folding, have been revealed. Nevertheless, research into the mechanisms and
control of the secretion pathways will lead to improved Bacillus protein
secretion systems and broaden the applications as industrial production host.
This review focuses on studies that aimed at optimizing B. subtilis as cell
factory for commercially interesting heterologous proteins.
Cui-Cui Miao et.al., studied onConstruction of a High-Expression System in
Bacillus through Transcriptomic Profiling and Promoter Engineering. Bacillus
subtilis is an ideal host for secretion and expression of foreign proteins. The
promoter is one of the most important elements to facilitate the high-level
production of recombinant protein. To expand the repertoire of strong promoters
for biotechnological applications in Bacillus species, 14 highly transcribed genes
based on transcriptome profiling of B. pumilus BA06 were selected and evaluated
for their promoter strength in B. subtilis. Consequently, a strong promoter P2069
was obtained, which could drive the genes encoding alkaline protease (aprE) and
green fluorescent protein (GFP) to express more eciency by an increase of 3.65-
fold and 18.40-fold in comparison with the control promoter (PaprE), respectively.
Further, promoter engineering was applied to P2069, leading to a mutation
promoter (P2069M) that could increase GFP expression by 3.67-fold over the wild-
type promoter (P2069). Moreover, the IPTG-inducible expression systems were
constructed using the lac operon based on the strong promoters of P2069 and
P2069M, which could work well both in B. Subtilis and B. pumilus. In this study,
highly ecient expression system for Bacillus was constructed based on
transcriptome data and promoter engineering, which provide not only a new option
for recombinant expression in B. subtilis, but also novel genetic tool for B.
pumilus.
Yuan Su et.al., studied on inherited backgrounds as well as simple and
diverse genetic manipulation systems, Bacillus subtilis is the key Gram-
positive model bacterium for studies on physiology and metabolism.
Furthermore, due to its highly efficient protein secretion system and
adaptable metabolism, it has been widely used as a cell factory for
microbial production of chemicals, enzymes, and antimicrobial materials
for industry, agriculture, and medicine. In this minireview, we first
summarize the basic genetic manipulation tools and expression systems for
this bacterium, including traditional methods and novel engineering
systems. Secondly, we briefly introduce its applications in the production
of chemicals and enzymes, and summarize its advantages, mainly focusing
on some noteworthy products and recent progress in the engineering of B.
subtilis. Finally, this review also covers applications such as microbial
additives and antimicrobials, as well as biofilm systems and spore
formation. We hope to provide an overview for novice researchers in this
area, offering them a better understanding of B. subtilis and its
applications.